Vol. 12, No. 5 Printed in US A.

INFECTION AND IMMUNITY, Nov. 1975, p. 1214-1218 Copyright (© 1975 American Society for Microbiology

Histopathological Effect of Clostridium perfringens Enterotoxin in the Rabbit Ileum J. L. McDONEL*

AND

C. L. DUNCAN

Food Research Institute and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 20 May 1975

Highly purified entertoxin from Clostridium perfringens was found to have histopathological activity in the rabbit ileum. Unlike the action of cholera, Escherichia coli, and Shigella enterotoxins, epithelium was denuded from the tips of ileal villi at concentrations of the enterotoxin necessary to induce fluid accumulation in the rabbit. Whether or not this observed histopathology is essential for the diarrheal syndrome associated with Clostridium perfringens food poisoning remains unclear.

The physiology and histopathology of experimental diarrhea induced in the rat by Clostridium perfringens recently has been studied and described (8, 9). One aspect of the pathology seen, villus epithelial desquamation, is important both in understanding the mode of action of the enterotoxin and comparing it to other enterotoxins currently under study (cholera, Escherichia coli, staphylococcal, and Shigella enterotoxins). Duncan et al. (1) first reported epithelial damage in rabbits in association with diarrhea induced by whole cells of enteropathogenic strains of C. perfringens, whereas Hauschild et al. (5) reported no significant lesions in lambs challenged by cells or culture filtrates. Niilo (11, 12) then reported that intravenous administration ofcrude cell extracts from sporulating cells of enteropathogenic strains of C. perfringens into lambs caused partial loss of villus epithelium in association with diarrhea and other systemic reactions. He also reported variable damage in ligated loops of lambs when the enterotoxin was placed in the lumen of the intestine. Some sections showed sloughing of epithelial cells and some did not, depending upon the dosages and the susceptibility of each animal. Rabbits that developed diarrhea after intravenous injection with crude cell extracts displayed an intact villus epithelial membrane. Guinea pigs treated similarly to the rabbits also showed intact epithelium but did not develop diarrhea. McDonel (8) showed that diarrhea in the rat due to highly purified C. perfringens enterotoxin is associated with epithelial desquamation, with the degree of damage being relative to the dosage of enterotoxin. Though diarrhea

could be developed with low doses of enterotoxin that caused only slight damage to the epithelium (8, 9), it is uncertain if damage is an essential part of the mechanism of diarrhea in the case of this enterotoxin. In the present study ileal loops of five adult white New Zealand rabbits were exposed in duplicate to various dosages of purified enterotoxin (16) for histology studies and four rabbits were treated similarly for fluid transport and protein determinations. After anesthesia with sodium pentobarbital (Nembutal) and subsequent cannulation and washing of the ileum with warm oxygenated Ringer glucose solution (8), loops were tied in 4- to 5-cm sections and excess fluid was removed by withdrawal with a syringe. Duplicate loops were then filled with 2 ml of Ringer solution containing 1,000, 500, 250, 100, or 50 erythemal units (EU) of enterotoxin. Controls consisted of loops containing 2 ml of solution without enterotoxin and untreated portions of intestine. The loops were carefully placed back into the abdominal cavity and the animal was kept covered and warm throughout the 90-min incubation period. At timed intervals the loops were removed, cut open lengthwise, and placed into phosphate buffered 4% formaldehyde solution prior to paraffin embedding and thin sectioning. Sections were stained with hematoxylin and eosin and with mucin stains. Fluid contents were withdrawn from loops prior to removal from rabbits studied for fluid production and protein release. The volumes were measured and total protein was determined from trichloroacetic acid precipitates by the method of Lowry et al. (7). All doses of enterotoxin caused some degree

FIG. 1. Histopathological effect of C. perfringens enterotoxin in the rabbit ileum. (A) Control; (B) portion of ileum exposed to 50 EU for 90 min; (C) 100 EU; (D) 250 EU; (E) 500 EU; (F) 1 ,000 EU. Magnification x100X. Strain, hematoxylin, and eosin. 1214

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VOL. 12, 1975

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of damage to the intestinal tissue in each sac TABLE 1. Fluid transport in rabbit ileal loops studied. Although variations in response did exposed to different doses of Clostridium perfringens enterotoxina occur, the severity of reaction increased with the dose in each animal. The sections shown in Total fluid Fig. 1 are representative of the damage at each Standard transport Erythemal activnb dosage. It was found that the response was dose ity/loop (jA/cm of intes- error Y tine)c dependent up to 500 EU, beyond which excess enterotoxin had no noticeable augmenting ef±15.9 7 0 (Control) 72.9 fect upon the histopathology described. Figure ±22.1 -50.1 8 50 1A shows control tissue exposed to solution 100 ±7.0 7 -40.0 without enterotoxin. It appeared normal, as did ±16.6 -61.8 8 250 untreated sections of the ileum. Figure 1B ±9.2 -57.7 8 500 shows slight but noticeable epithelial damage ±25.5 3 -66.7 1,000 with exposure to 50 EU of enterotoxin. Cona All loops were exposed for 90 min. gested submucosa was also evident (not bn, Number of loops. shown). In Fig. 1C (100 EU) a few epithelial c cells are present in the lumen from the par- men.Negative values indicate secretion into the lutially desquamated epithelium. Mucin was also present that had been expelled from the globlet TABLE 2. Protein content offluid from rabbit ileal cells. Figure 1D (250 EU) is progressive and loops exposed to Clostridiumperfringens enterotoxina shows further desquamation. Intense desquaErythemal activ- Total protein Standard ernb mation, inflamatory cells (mostly lymphoror Y (mg/cm) ity/loop cytes), and slight hyperemia in the mucosa are seen in Fig. 1E (500 EU). Figure 1F (1,000 EU) 0.03 8 0 (control) 0.31 shows a degree of damage similar to that in 0.10 8 50 1.46 7 100 0.13 1.79 Fig. 1E. 0.33 6 2.15 250 It can be seen (Table 1) that net fluid accumu2.24 0.12 7 500 lation in the lumen was the characteristic re2.20 0.55 3 1,000 sponse to the enterotoxin as compared to a decrease in fluid content in control loops. Each a All loops were exposed for 90 min. dose of toxin caused a significant difference bn, Number of loops. from controls in fluid transport (P < 0.001) as determined by Student's t test. However, there activity. Hauschild et al., by using a rapid dewas no significant difference between amounts tection procedure using a 90-min incubation of fluid secreted at the doses studied. This sug- period (4), found that as little as 2.5 EU of gests a possible threshold of response for fluid enterotoxin was sufficient to prevent absorptransport above which excess stimulation has tion of fluid whereas between 40 and 160 EU little or no augmenting effect. That, however, was needed to cause a net increase in fluid is not the case for tissue damage. The data in volume of loops tested. It can be seen from the Table 2 support those seen in Fig. 1, in that evidence presented here that the minimal luminal fluid protein content increased with range of toxin needed to induce fluid production the toxin dose. This would be expected since is within the range needed to induce tissue increased desquamation seen with increased damage in the rabbit. This report establishes that a distinct histopadosages (Fig. 1) and subsequent leakage of fluids into the lumen would cause an increase thology is produced in the rabbit intestine by in protein to be found there. The differences the intraluminal administration of C. perfrinbetween protein release induced by 50 and 250 gens enterotoxin at doses needed to induce fluid EU and by 100 and 500 EU were significant (P accumulation in ileal loops. This is similar to results obtained in the rat (8). How the desqua< 0.05). It seems that an increase of nearly 200 EU was needed at the time interval studied to mation of villus epithelium is related to the induce a significant increase in fluid protein transport reversals associated with diarrhea content. The addition of more than 500 EU due to this enterotoxin is not yet clear. Certainly if tissue disruption is a part of the patholcaused no significant increase in protein. Stark and Duncan (16) found the minimal ogy in human cases it would contribute to fluid dose of enterotoxin needed to induce measura- and electrolyte loss. However, it would not be ble fluid accumulation by the rabbit ileal loop expected that the damage in human cases could test, as measured after an incubation period of be very severe as apparent recovery is often several hours, to be about 140 to 200 EU of complete in 24 h. The susceptibility of human

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epithelial tissue to the desquamating activity of the enterotoxin could be very different from that seen in these experimental models. The action of C. perfringens enterotoxin in the rabbit model is in contrast to that of cholera toxin in the rabbit (13), which causes no apparent change in the villus epithelium. Cholera toxin does not cause noticeable damage in the canine model (2) or human cases of the disease (3). E. coli enterotoxin also acts very similarly to cholera toxin in the rabbit (10) and therefore is contrasted to this enterotoxin. Although Shigella enterotoxin alone has no effect upon villus morphology (6), staphylococcal enterotoxin has been shown to be capable of denuding villus epithelium in cats (15) and dogs (12).

INFECT. IMMUN.

LITERATURE CITED 1. Duncan, C. L., H. Sugiyama, and D. H. Strong. 1968.

tinal morphology in human and experimental cholera. Arch. Pathol. 77:529-537. 4. Hauschild, A. H. W., R. Hilsheimer, and C. G. Rogers. 1971. Rapid detection of Clostridium perfringens enterotoxin by a modified ligated intestinal loop technique in rabbits. Can. J. Microbiol. 17:1475-1476. 5. Hauschild, A. H. W., L. Niilo, and J. Dorward. 1967. Experimental enteritis with food poisoning and classical strains of Clostridium perfringens type A in lambs. J. Infect. Dis. 117:379-386. 6. Levine, M. M., H. L. DuPont, S. B. Formal, R. B. Hornick, A. Takevchi, E. J. Gangarosa, M. J. Snyder, and J. P. Libonati. 1973. Pathogenesis of Shigella dysenteriae 1 (Shiga) dysentery. J. Infect. Dis. 127:261-270. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 8. McDonel, J. L. 1974. In vivo effects of Clostridium perfringens enteropathogenic factors on the rat ileum. Infect. Immun. 10:1156-1162. 9. McDonel, J. L., and T. Asano. 1975. Analysis of unidirectional fluxes of sodium during diarrhea induced by Clostridium perfringens enterotoxin in the rat terminal ileum. Infect. Immun. 11:526-529. 10. Moon, H. W., S. C. Whipp, and A. L. Baetz. 1971. Comparative effects of enterotoxins from Escherichia coli and Vibrio cholerae on rabbit and swine small intestine. Lab. Invest. 25:133-140. 11. Niilo, L. 1970. Mechanism of action of the enteropathogenic factor of Clostridium perfringens type A. Infect. Immun. 3:100-106. 12. Niilo, L. 1973. Effect on calves of the intravenous injection of the enterotoxin of Clostridium welchii Type A. J. Comp. Pathol. 83:265-269. 13. Norris, H. T., and G. Majno. 1968. On the role of the ileal epithelium in the pathogenesis of experimental cholera. Am. J. Pathol. 53:263-280. 14. Prohaska, J. V. 1963. Role of staphylococcal enterotoxin in the induction of experimental ileitis. Ann.

Rabbit ileal loop response to strains of Clostridium perfringens. J. Bacteriol. 95:1560-1566. 2. Elliott, H. L., C. C. J. Carpenter, Jr., R. B. Sack, and J. H. Yardley. 1970. Small bowel morphology in experimental canine cholera. A light and electron microscopic study. Lab. Invest. 22:112-120. 3. Fresh, J. W., P. M. Versage, and F. Reyes. 1964. Intes-

Surg. 158:492497. 15. Prohaska, J. V., M. J. Jacobson, C. T. Drake, and T. Tan. 1959. Staphylococcus enterotoxin enteritis. Surg. Gynecol. Obstet. 109:73-77. 16. Stark, R. L., and C. L. Duncan. 1972. Purification and biochemical properties of Clostridium perfringens type A enterotoxin. Infect. Immun. 6:662-673.

This research was supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Public Health Service research grant AI-11865-05 from the National Institute of Allergy and Infectious Diseases, Public Health Service research grant FD-00203-05 from the Food and Drug Administration, and by Contributors to the Food Research Institute by member industries. J.L.M. is the recipient of a post-doctoral award from Public Health Service grant T32-EF0715-01 of the National Institute of Environmental Health Sciences. C.L.D. is the recipient of a Public Health Service Research Career Development award AI-70721-02 from the National Institute of Allergy and Infectious Diseases. We thank Joseph Lalich for assistance in interpreting histopathological data.

Histopathological effect of Clostridium perfringens enterotoxin in the rabbit ileum.

Vol. 12, No. 5 Printed in US A. INFECTION AND IMMUNITY, Nov. 1975, p. 1214-1218 Copyright (© 1975 American Society for Microbiology Histopathologica...
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